Inhibition of CBP/β-catenin signaling ameliorated fibrosis in cholestatic liver disease

Abstract

Chronic cholestatic liver diseases are characterized by injury of the bile ducts and hepatocytes caused by accumulated bile acids (BAs) and inflammation. Wnt/β-catenin signaling is implicated in organ fibrosis; however, its role in cholestatic liver fibrosis remains unclear. Therefore, we explored the effect of a selective cAMP response element-binding protein-binding protein (CBP)/β-catenin inhibitor, PRI-724, on murine cholestatic liver fibrosis. PRI-724 suppressed liver fibrosis induced by multidrug resistance protein 2 knockout (KO), bile duct ligation, or a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet; it also suppressed BA synthesis and macrophage infiltration. The expression of early growth response-1 (Egr-1), which plays a key role in BA synthesis, was increased in the hepatocytes of patients with cholestatic liver disease. PRI-724 inhibited Egr-1 expression induced by cholestasis, and adenoviral shEgr-1-mediated Egr-1 knockdown suppressed BA synthesis and fibrosis in DDC diet-fed mice, suggesting that PRI-724 exerts its effects, at least in part, by suppressing Egr-1 expression in hepatocytes. Hepatocyte-specific CBP KO in mice suppressed BA synthesis, liver injury, and fibrosis, whereas hepatocyte-specific KO of P300, a CBP homolog, exacerbated DDC-induced fibrosis. Intrahepatic Egr-1 expression was also decreased in hepatocyte-specific CBP-KO mice and increased in P300-KO mice, indicating that Egr-1 is located downstream of CBP/β-catenin signaling. Conclusion: PRI-724 inhibits cholestatic liver injury and fibrosis by inhibiting BA synthesis in hepatocytes. These results highlight the therapeutic effect of CBP/β-catenin inhibition in cholestatic liver diseases.

FIGURE 1

PRI‐724 suppresses liver injury and fibrosis in multidrug resistance protein 2 (Mdr2)–knockout (KO) mice. Male Mdr2‐KO mice (8–10 weeks old, n = 12) and friend leukemia virus B (FVB) background control mice (n = 6) were treated or not with PRI‐724 (20 mg/kg, 3 times a week) for 10 weeks. (A) Scheme of the treatment protocol. (B–D) Collagen deposition as assessed by sirius red staining (B; scale bars, 500 μm, 1 mm, and 500 μm, respectively, from the top figure) and graph (C) and by measuring hydroxyproline contents (D). (E) Serum matrix metalloproteinase (MMP) levels as analyzed using a Bio‐Plex assay. (F) Messenger RNA (mRNA) expression of the indicated genes in the livers as determined by real‐time quantitative polymerase chain reaction (PCR). (G,H) Hematoxylin and eosin (HE) staining and immunohistochemical staining using anti‐CK19 and Ki67 antibodies (scale bars, 100 μm, 250 μm, and 50 μm, respectively, from the top figure) (G) and quantification of the positive cells (H). (I) Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (T.Bil) levels (n = 5–7 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001. Abbreviations: Col1A1, collagen type I alpha 1; i.p., intraperitoneally; n.s., not significant versus control‐treated Mdr2‐KO mice by one‐way analysis of variance; TIMP1, tissue inhibitor of metalloproteinase 1.

FIGURE 2

PRI‐724 reduces bile acid (BA) synthesis in Mdr2‐KO mice. Male Mdr2‐KO mice (8–10 weeks old, n = 12) and FVB background control mice (n = 6) were treated or not with PRI‐724 (20 mg/kg, 3 times a week) for 10 weeks. (A) Immunohistochemical staining using anti‐S100A4, F4/80, and Ki67 antibodies (scale bars, 250 μm). (B) Quantification of S100A4‐positive and F4/80‐positive cells and GS‐positive areas in the livers. (C) Clustergrams of PCR array analyses of WNT/β‐catenin‐related genes (left panel, WNT/β‐catenin signals; right panel, WNT/β‐catenin targets). (D) Early growth response‐1 (Egr‐1) mRNA expression in the livers as determined by real‐time quantitative PCR (n = 10 per group). (E) Immunohistochemical staining using anti‐Egr‐1 antibodies (scale bar, 100 μm). (F) Quantification of Egr‐1‐positive cells and glutamine synthetase (GS)–positive areas in the livers. (G) Hepatic and serum total BA (TBA), cholic acid (CA), and chenodeoxycholic acid (CDCA) levels (n = 10 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001, by Student's t test.

FIGURE 3

PRI‐724 suppresses liver injury and fibrosis accompanied by reduced hepatic and serum BA levels. Male wild‐type C57BL/6 male 8–10‐week‐old mice were subjected to bile duct ligation (BDL) or sham operation and treated with PRI‐724 (20 mg/kg, 3 times a week) or phosphate‐buffered saline (PBS). The animals were killed on day 14 after surgery. (A) Scheme of the treatment protocol. (B–D) HE, sirius red staining, and immunohistochemical staining for CK19 (B; scale bars, 100, 500, and 250 μm, respectively, from the top figure). Quantification of the sirius red–positive area (C) and CK19‐positive cells (D). (E) Collagen deposition as assessed by measuring hydroxyproline contents. (F) Serum ALT, ALP, and T.Bil levels. (G) Clustergrams of PCR array analyses of WNT/β‐catenin‐related genes (left panel, WNT/β‐catenin signals; right panel, WNT/β‐catenin targets). (H) Egr‐1 mRNA expression in the livers as determined by real‐time quantitative PCR (n = 3–12 per group). (I) Immunohistochemical staining using anti‐Egr‐1 antibodies (scale bar, 50 μm). (J) Quantification of Egr‐1‐positive cells in the livers. (K) Hepatic TBA, CA, and CDCA levels (n = 6–9 per group). The results shown are representative of at least three independent experiments. Data represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 by one‐way ANOVA (C–F,H,K) or unpaired Student's t test (J).

FIGURE 4

PRI‐724 suppresses liver injury and 3.5‐diethoxycarbonyl‐1.4‐dihydrocollidine (DDC) diet–induced fibrosis. Eight‐week‐old wild‐type C57BL/6 male mice were fed a 0.1% DDC diet for 18 days and administered PRI‐724 (20 mg/kg, 3 times a week) or PBS. (A) HE and sirius red staining (scale bars, 250 and 500 μm, respectively, from the top figure). (B) Collagen deposition as assessed by hydroxyproline measurement (n = 12 per group). (C,E) Messenger RNA (mRNA) expression of the indicated genes in the livers as determined by quantitative reverse‐transcription PCR (n = 12 per group). (D,F) Immunohistochemical staining for Egr‐1 (scale bars, 50 μm). (G) Quantification of Egr‐1‐positive cells in the livers. (H) Hepatic and serum TBA, CA, and CDCA levels (n = 6 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.005 by unpaired Student's t test.

FIGURE 5

Liver fibrosis outcomes differ between cAMP response element‐binding protein‐binding protein (CBP) and P300‐Alb/Cre KO mice on a DDC diet. Alb/Cre‐CBP‐KO, Alb/Cre‐P300KO, and control Alb/Cre male mice (8 weeks old, n = 15 per group) were fed a 0.1% DDC diet for 18 days. (A) Scheme of the treatment protocol. (B) HE, sirius red, and immunohistochemical staining for CK19 and Ki67 (scale bars, 250, 250, 500, 100, and 50 μm, respectively, from the top figure). (C) Quantification of CK19‐positive and Ki67‐positive in the liver. (D) Collagen deposition as assessed by hydroxyproline measurement. (E) mRNA expression of the indicated genes in the livers as determined by real‐time quantitative PCR (n = 10 per group). (F) Serum ALT, ALP, and T.Bil levels (n = 10–15 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 by one‐way ANOVA.

FIGURE 6

CBP KO in hepatocytes results in reduced hepatic and serum BA levels and decreased Egr‐1 expression. Alb/Cre‐CBP‐KO, Alb/Cre‐P300KO, and control Alb/Cre male mice (8 weeks old, n = 15 per group) were fed a 0.1% DDC diet for 18 days. (A) Immunohistochemical staining for S100A4, F4/80, CBP, P300, and GS (scale bars, 50 μm, except for P300 and GS [100 μm]). (B) Quantification of S100A4‐positive and F4/80‐positive cells and GS‐positive areas in the livers. (C) Egr‐1 mRNA expression in the livers as determined by real‐time quantitative PCR (n = 15 per group). (D) Immunohistochemical staining using anti‐Egr‐1 antibodies (scale bar, 50 μm). (E) Quantification of Egr‐1‐positive cells in the livers (n = 6). (F) Hepatic TBA, CA, and CDCA levels (n = 6 per group). The results shown are representative of at least three independent experiments. (G) mRNA expression of the indicated BA metabolism–related genes in the livers as determined by real‐time quantitative PCR (n = 15 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 by one‐way ANOVA.

FIGURE 7

Egr‐1 knockdown inhibits liver injury and fibrosis induced by DDC. Male wild‐type C57BL/6 mice (8 weeks old) were fed a 0.1% DDC diet for 18 days. The mice received a single injection of adeno‐associated virus 8 (AAV8)/shEgr‐1 and AAV8/control (n = 5 in each group) at a dose of 2 × 10¹² VG/mouse 4 days after the start of the DDC diet. (A) Scheme of the treatment protocol. (B) HE, sirius red, and immunohistochemical staining for Egr‐1 (scale bars, 100, 250, and 100 μm, respectively, from the top figure) (n = 5). (C) Collagen deposition as assessed by hydroxyproline measurement (n = 5 per group). (D,E,H) mRNA expression of the indicated genes in the livers as determined by real‐time quantitative PCR (n = 5 per group). (F) Serum ALT, ALP, and T.Bil levels (n = 5 per group). (G) Hepatic TBA, CA, and CDCA levels (n = 5 per group). The results shown are representative of at least three independent experiments. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 by unpaired Student's t test. Abbreviations: BSEP, bile salt export pump; FXR, farnesoid X receptor; NTCP, sodium taurocholate cotransporting polypeptide.

FIGURE 8

Egr‐1 expression is enhanced in human cholestatic liver disease. (A) Immunohistochemical staining for CBP, P300, and Egr‐1 in liver biopsy tissues from patients with metastatic liver tumors (non‐cancerous tissues) and patients with primary biliary cholangitis (PBC). Scale bars, 50 μm. (B) Quantification of Egr‐1‐positive hepatocytes. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001 by an unpaired Student's t test.